title: Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage.
abstract: Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.
authors.affiliation: Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
authors.forename: Sergey
authors.initials: S
authors.lastname: Alekseev
authors.forename: Martijn S
authors.initials: MS
authors.lastname: Luijsterburg
authors.forename: Alex
authors.initials: A
authors.lastname: Pines
authors.forename: Bart
authors.initials: B
authors.lastname: Geverts
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Mari
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Giglia-Mari
authors.forename: Hannes
authors.initials: H
authors.lastname: Lans
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
authors.forename: Leon H F
authors.initials: LH
authors.lastname: Mullenders
authors.forename: Jan H J
authors.initials: JH
authors.lastname: Hoeijmakers
authors.forename: Wim
authors.initials: W
authors.lastname: Vermeulen
mesh_terms: D000818:Animals; D002460:Cell Line; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: C114234:CUL4A protein, human; D044842:Cullin Proteins; C091435:DDB1 protein, human; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C086984:XPC protein, human; D004247:DNA